Transcriptome analysis of follicular granulosa cells in young women with diminished ovarian reserve.

Abstract

Background: Diminished ovarian reserve (DOR) is defined as the decline in both quantity and quality of oocytes in the ovarian cortex. In recent years, DOR has shown an increasing trend among young women. However, the specific etiology and detailed pathogenesis in this population remain unclear. Granulosa cells (GCs) play crucial roles in oocyte growth, development, energy absorption, and transcriptional regulation. This study aimed to investigate the differential expression profiles of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs) in GCs from young women with DOR, and to explore the potential molecular mechanisms through competing endogenous RNA (ceRNA) network analysis.

Methods: A total of 80 young women (≤ 35 years) undergoing in vitro fertilization-embryo transfer (IVF-ET) were prospectively enrolled between April 2024 and December 2024, including 40 with normal ovarian reserve (NOR) and 40 with DOR. Granulosa cells were collected during oocyte retrieval. High-throughput sequencing was performed to identify differentially expressed miRNAs, lncRNAs, and mRNAs. Bioinformatics analysis including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was conducted. The ceRNA regulatory network was constructed using Cytoscape. Quantitative real-time PCR (qRT-PCR) was used to validate the sequencing results.

Results: Compared with the NOR group, young women with DOR showed significantly higher basal follicle-stimulating hormone (FSH) levels, and significantly lower anti-Müllerian hormone (AMH) levels and antral follicle count (AFC) (all P < 0.05). The DOR group also had significantly lower numbers of retrieved oocytes, metaphase II oocytes, two-pronuclear zygotes, and day 3 available embryos (all P < 0.05). A total of 36 differentially expressed miRNAs (5 upregulated, 31 downregulated), 102 differentially expressed lncRNAs (55 upregulated, 47 downregulated), and 234 differentially expressed mRNAs (69 upregulated, 165 downregulated) were identified. Functional enrichment analysis revealed that these differentially expressed RNAs were mainly involved in fatty acid metabolism, AKT-mTOR signaling pathway, apoptosis, DNA damage and repair, and steroidogenesis. Bioinformatics analysis predicted a ceRNA network containing 20 lncRNA-mRNA pairs, 108 lncRNA-miRNA pairs, and 89 miRNA-mRNA pairs. Two potential ceRNA axes were identified by qRT-PCR-based expression validation: lncRNA MSTRG.39605.16/miR-143-5p/FADS1 and lncRNA NONHSAT208904.1/miR-199b-3p/ACP3, both associated with fatty acid metabolism regulation. However, direct molecular interactions within these axes remain to be functionally validated."

Conclusions: Transcriptional alterations in granulosa cells from young women with DOR are primarily enriched in fatty acid metabolism, AKT-mTOR signaling, and apoptosis pathways. The predicted ceRNA networks represent hypothetical regulatory mechanisms that may potentially be involved in DOR pathogenesis through modulation of fatty acid metabolic processes, pending functional validation. These findings provide candidate molecular markers and potential targets for future mechanistic studies of this condition.